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Thin Layer Chromatography

JOSEPH SHERMA
Department of Chemistry, Lafayette College, Easton, PA, USA

I.

INTRODUCTION

Thin layer chromatography (TLC) is a type of liquid chromatography in which the stationary phase is in the form of a layer on a glass, an aluminum, or a plastic support. The term planar chromatography is often used for both TLC and paper chromatography (PC) because each employs a planar stationary phase rather than a packed column. PC, which utilizes plain, modied, or impregnated paper (cellulose) as the stationary phase, involves many of the same basic techniques as TLC, but it has not evolved into an efcient, sensitive, quantitative, instrument-based analytical method and has many disadvantages relative to TLC. Consequently, PC will not be covered in this chapter. As originally developed in 1951 by J.G. Kirchner and colleagues, later standardized by E. Stahl and colleagues, and still widely practiced today (Fried and Sherma, 1999a; Sherma, 2002), classical, capillary-action TLC is an inexpensive, easy technique that requires little instrumentation, which is used for separation of simple mixtures and for qualitative identication or semiquantitative, visual analysis of samples. In contrast, modern TLC [usually termed as high performance thin layer chromatography (HPTLC)], which began around 1975 with the introduction of high efciency, commercially precoated plates by Merck, is an instrumental technique carried out on efcient, ne-particle layers. Instrumental HPTLC is
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capable of producing fast, high-resolution separations and qualitative and quantitative results that meet good manufacturing practices (GMP) and good laboratory practices (GLP) standards. The accuracy and precision of data obtained by HPTLC rival those of gas chromatography (GC) and high performance column liquid chromatography (HPLC), and it has many advantages relative to these methods. TLC is an off-line process in which the various stages (Fig. 1) are carried out independently. The advantages of this arrangement using an open, disposable layer compared with an on-line column process such as HPLC include the possibility of separating up to 70 samples and standards simultaneously on a single plate, leading to high throughput, low cost analyses and the ability to construct calibration curves from standards chromatographed under the same conditions as the samples; analyzing samples with minimum sample preparation without fear of irreversible contamination of the column or carryover of fractions from one sample to another as can occur in HPLC with sequential injections; and analyzing a sample by use of multiple separation steps and static postchromatographic detection procedures with various universal and specic visualization reagents, which is possible because all sample components are stored on the layer without chance of loss. TLC is highly selective and exible because of the great variety of layers that is available commercially. It has proven to be as sensitive

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An apparent disadvantage of TLC is that although all of the individual steps have been automated and on-line coupling with other chromatographic and spectrometric techniques has been achieved, complete automation of TLC has not been realized. However, changing the offline nature of TLC to an on-line closed system with complete automation would eliminate many of the advantages stated earlier. Several studies have shown that more samples per day can be processed using stepwiseautomated TLC compared with a fully automated HPLC system, and with lower cost (Abjean, 1993). Analytical TLC differs from preparative layer chromatography (PLC) in that larger weights and volumes of samples are applied to thicker (0.5 2 mm) and sometimes larger layers in the latter method, the purpose of which is the isolation of 10 1000 mg of sample for further analysis. This chapter describes the TLC techniques and instrumentation with different levels of automation that can be used in a contemporary analytical laboratory to produce high-quality analytical results without sacricing the great exibility of the method.

II.

SAMPLE PREPARATION

Figure 1 Schematic diagram of the steps in a TLC analysis. (Courtesy of Camag.)

as HPLC in many analyses, and solvent usage per sample is extremely low. TLC and HPTLC plates are usually developed by capillary ow of the mobile phase without pressure in ascending or horizontal modes, but forced ow methods, in which the mobile phase is driven through the layer by pumping under pressure or by centrifugal force, are also used. In capillary-ow TLC, the migration speed of the mobile phase decreases with the square of the solvent migration distance. Limited separation efciency is a disadvantage of TLC. The maximum number of theoretical plates (N) for HPTLC is ca. 5000 compared with ca. 15,000 for HPLC, while the separation number (the number of spots that can be separated over the distance of the run with a resolution of unity) is ca. 15 in TLC compared with 200 in HPLC. This limited efciency is a result of the capillary-ow mechanism over a restricted migration distance. Under forced ow conditions, TLC separation efciency is signicantly improved. For a discussion on the theory and mechanism of the various modes of TLC, see Fried and Sherma (1999b), Kowalska and Prus (2001), and Kowalska et al. (2003).

Sample preparation (Fried and Sherma, 1999a, c; Sherma, 2003) procedures for TLC are similar to those for GC and HPLC. The solution to be spotted must be sufciently concentrated so that the analyte can be detected in the applied volume, and pure enough so that it can be separated as a discrete, compact spot or zone. The solvent in which the sample is dissolved must be suitable in terms of viscosity, volatility, ability to wet the layer, and potential for unintended predevelopment during sample application. Relatively pure samples or their concentrated extracts can often be directly spotted for TLC analysis. If the analyte is present in low concentration in a complex sample, solvent extraction, cleanup (purication), and concentration procedures must precede TLC. Because layers are not reused, it is often possible to apply cruder samples than could be injected into a GC or HPLC column, including samples with irreversibly sorbed impurities. However, impurities that comigrate with the analyte, adversely affect its detection, or distort its zone (i.e., cause streaking or tailing), must be removed prior to TLC. Carryover of material from one sample to another is not a problem as it is in on-line column methods involving sequential injections. Therefore, sample preparation is often simpler for TLC compared with other chromatographic methods. Common cleanup procedures include liquid liquid extraction, column chromatography, desalting, and deproteinization. Solid-phase extraction (SPE) using small,

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Figure 2 J.T. Baker vacuum processor for 12 or 24 BAKERBOND SPE or Speedisk columns. (Courtesy of Mallinckrodt Baker Inc., Phillipsburg, NJ.)

disposable columns and a manual vacuum manifold (Fig. 2) or various semiautomated or automated instruments has become widely used for isolation and cleanup of samples prior to TLC analysis, such as pesticide residues in water (Hamada and Wintersteiger, 2002). Supercritical uid extraction has been used for off-line extraction of analytes from samples (van Beek, 2002), and has been directly coupled to TLC for analysis of solid samples or solutions loaded on glass ber lters (Esser and Klockow, 1994). A derivative of the analyte can be formed in solution prior to spotting, or in situ at the origin by overspotting of a reagent, in order to improve resolution or detection. Special plates with preadsorbent zone serve for sample cleanup by retaining some interfering substances. III. STATIONARY PHASES

increased resolution of zones is offset by diffusion effects. In comparison with TLC, HPTLC provides better separation efciency and lower detection limits. The choice of the layer and mobile phase is made in relation to the nature of the sample. Normal-phase (NP) or straight-phase adsorption TLC on silica gel with a less polar mobile phase, such as chloroform methanol, is the most widely used mode. Lipophilic C-18, C-8, and C-2 bonded silica gel phases with a polar aqueous mobile phase, such as methanol water, are used for reversedphase (RP) TLC. Other precoated layers include alumina, magnesium silicate (Florisil), polyamide, cellulose, ion exchangers, and chemically bonded phenyl, amino, cyano, and diol layers. The latter three bonded phases can function with multimodal mechanisms, depending on the composition of the mobile phase. Silica gel can be impregnated with various solvents, buffers, and selective reagents to improve separations. Chiral plates composed of a RP layer impregnated with copper acetate and a chiral selector, (2S,4R,20 RS)-4-hydroxy-1-(2-hydroxydodecyl)proline, can be used to separate enantiomers through a ligand-exchange mechanism. Preparative layers are thicker than analytical layers to provide higher sample capacity. Ultra-thin silica layers (Hauck et al., 2002) are the newest type available commercially. Unlike all other TLC and HPTLC layers, these do not consist of particulate material but are characterized by a monolithic silica structure. They are manufactured without a binder, which is usually needed to stabilize the sorbent particles on the support, and have a signicantly thinner layer (10 mm), leading to short migration distances, fast development times, and very low solvent consumption. Important manufacturers of TLC plates include Merck, Whatman, Analtech, and Macherey-Nagel, and literature from these companies should be consulted for details of availability, properties, usage, and applications.

IV. MOBILE PHASES Unlike GC, in which the mobile phase (carrier gas) is not a factor in the selectivity of the chromatographic system, the mobile phase in liquid chromatography, including TLC, exerts a decisive inuence on the separation. In HPLC, the analyte passes through the on-line detector in the presence of the mobile phase. Therefore, solvents must be chosen not only to provide the required resolution, but also to not absorb at the ultraviolet (UV) detection wavelength. Because in TLC the mobile phase is removed (evaporated) before the zones are detected, a wider variety of solvents can be used to prepare mobile phases compared with HPLC.

TLC and HPTLC plates are commercially available in the form of precoated layers supported on glass, plastic sheets, or aluminum foil (Fried and Sherma, 1999d; Lepri and Cincinelli, 2001; Rabel, 2003). HPTLC plates are smaller (10 10 or 10 20 cm), have a thinner (0.1 0.2 mm) more uniform layer composed of smaller diameter particles (5 6 mm), and are developed over shorter distances (ca. 3 7 cm) compared with classical 20 20 cm TLC plates, which have a 0.25 mm thick layer of 12 20 mm particle size and are developed for 10 12 cm. Optimal development distances are the points beyond which

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The mobile phase (Fried and Sherma, 1999e) is usually a mixture of two to ve different solvents selected empirically using trial and error guided by prior personal experience and literature reports of similar separations. In addition, various systematic mobile phase optimization approaches (Cimpoiu, 2003; Prus and Kowalska, 2001) involving solvent classication (selectivity) and the elutropic series (strength) patterned after HPLC have been described, most notably, the PRISMA model based on Snyders solvent classication system as developed by Nyiredy. For NP-TLC (silica gel), the following 10 solvents from Snyders eight selectivity groups are used for exploratory TLC of the mixture: diethyl ether (group I), isopropanol and ethanol (group II), tetrahydrofuran (group III), acetic acid (group IV), dichloromethane (group V), ethyl acetate and dioxane (group VI), toluene (group VII), and chloroform (group VIII). Hexane (solvent strength 0) is used to adjust the Rf values within the optimum range (0.20.8), if necessary. Between two and ve solvents are then selected for construction of the PRISMA model that leads to identication of the optimized mobile phase. A similar procedure is followed for RP-TLC (e.g., C-18 bonded phase layer) using mixtures of methanol, acetonitrile, and/or tetrahydrofuran with water (solvent strength 0). TLC is usually carried out with a single mobile phase, rather than a mobile phase gradient as is often used in HPLC. Equilibration between the mobile phase and the layer occurs gradually during TLC development, and the mobile phase composition can change because different constituents migrate through the layer at different rates (solvent demixing). This leads to solvent gradients along the layer during isocratic TLC, the formation process of which is very different than the intentional, wellcontrolled gradients in a fully equilibrated HPLC column. Mobile phase selection for automated multiple development (AMD) is described in Section VI,A,4.

TLC, 0.5 5 mL volumes are usually applied manually with a micropipet to produce initial zones with diameters in the 2 4 mm range. For TLC or HPTLC, initial zones in the form of spots can be applied from a disposable 0.5, 1, 2, or 5 mL xed-volume, seloading glass capillary pipet, which is held in a rocker-type spotting device (the Nanomat 4, Camag Scientic Inc., Wilmington, NC) that mechanically controls its positioning and brings the capillary tip in gentle and uniform contact with the layer to discharge the solution without damage to the layer. The Nanomat and other instruments for sample application and the later steps in the TLC process have been described by Reich (2003). The TLS100 (Baron, Reichenau, Germany) is an automated apparatus for applying spots of up to 30 samples and four standards using a 1, 10, or 100 mL motor driven syringe. Locations and volumes can be chosen with a keypad for application onto as many as six HPTLC plates. Sample application in the form of bands is advantageous for high-resolution separations of complex samples, improved detection limits, and for precise [1% relative standard deviation (RSD)] quantitative scanning densitometry using the aliquot technique (scanning with a slit of one-half to two-thirds the length of the applied band). Narrow, homogeneous sample bands of controlled length [from 1 (spot) to 195 mm] can be applied by use of a spray-on device (the Camag Linomat 5, Fig. 3), in which the plate is mechanically moved right to left in the X-direction beneath a xed syringe from which

V. APPLICATION OF SAMPLES Application of small, exactly positioned initial zones of sample and standard solutions (Fried and Sherma, 1999f) having accurate and precise volumes, without damaging the layer surface, is critical for achieving maximum resolution and reliable qualitative and quantitative analysis. The volumes applied and the method of application depend on the type of analysis to be performed (qualitative or quantitative), the layer (TLC or HPTLC), and the detection limit. For the greatest separation efciency, the solvent in which the sample is dissolved should have high volatility and be as low in solvent strength as possible (nonpolar for NP systems and polar for RP systems) to retard the possibility of prechromatography during application. Application of round spots allows the maximum number of samples to be applied onto a given plate. For
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Figure 3

Linomat 5. (Courtesy of Camag.)

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0.1 2000 mL of sample is sprayed by an atomizer operating with a controlled nitrogen gas pressure for analytical and preparative applications. The user selects the sample volumes and Y-position via a keypad or by downloading a method from a personal computer, and the instrument exactly positions the initial zones, which facilitates automated scanning after chromatogram development and overspraying samples with a reagent for in situ prechromatographic derivatization or with spiking solutions for validation of quantitative analysis by the standard-addition method. With the correct choice of application parameters, less volatile and higher strength sample solvents can be tolerated without forming broadened initial zones. The ability to apply larger volumes to an HPTLC plate without loss of resolution lowers the determination limits with respect to the concentration of the solution, which aids in trace analysis. Complex, impure samples can often be successfully quantied only if bands are applied rather than spots. The Linomat facilitates quantitative analysis by allowing different volumes of the same standard solution to be applied to produce the densitometric calibration curve, rather than the same volume of a series of standards when spots are applied. The AS30 (Desaga, Weisloch, Germany) is a software controlled, fully automated band or spot applicator that also works according to a spray-on technique, in which a stream of gas carries the sample from the cannula tip onto the plate. The syringe does not have to be manually lled by the user, as with the Linomat. During the lling process, the dosing syringe is positioned over the tray, which collects rinsing and ushing solvent and excess sample. The sample is injected into the body of the syringe through a lateral opening. After the syringe has been lled, a stepping motor moves the piston downwards to dose the llport. A second stepping motor moves the tower sideways across the plate. The microprocessor controls both motors and the gas valve for accurate and precise application in the form of spots or bands. All parameters for application of up to 30 samples are entered via the keyboard. The user is guided through the clearly structured menu by the two-line LCD display. Figure 4 shows the Camag Automatic TLC Sampler IV (ATS 4), which is an advanced, fully automated, computer-controlled device for sequential application of up to 66 samples from a rack of vials or 96 samples from well plates through a steel capillary as spots by contact transfer or as bands by the spray-on technique. The speed, volume, and X- and Y-position pattern of application are controllable, and a programmable rinse cycle can eliminate cross-contamination. Low concentration samples can be applied as rectangles, which are focused into narrow bands by predevelopment with a strong mobile phase. An optional heated spray nozzle allows increased application speed, which is important for aqueous solutions. Analyses performed with this
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Figure 4 Camag.)

Automatic TLC Sampler 4 (ATS 4). (Courtesy of

applicator combined with densitometric chromatogram evaluation controlled by the same computer conform to GMP/GLP standards.

VI.

CHROMATOGRAM DEVELOPMENT

TLC is almost always carried out in the elution mode. Methods and applications of displacement TLC have been described (Bariska et al., 2000), but this method is not yet widely used and will not be covered in this chapter. Electro-osmotically driven TLC (Nurok et al., 2002) is a quite new method that has not yet been shown to have a signicant number of important practical applications, and it also will not be covered. Linear development of TLC plates is used almost exclusively today. Therefore, circular (radial) and anticircular development will not be discussed here. TLC development times are typically in the range of 360 min, depending on the layer, mobile phase, and development method chosen. However, the development time does not signicantly inuence the overall analysis time per sample, because many samples and standards can be chromatographed simultaneously. Development modes and chambers have been described in detail elsewhere (Fried and Sherma, 1999g). A. 1. Capillary-Flow TLC Ascending Development

The results of TLC are strongly dependent upon the environmental conditions during development, such as small changes in mobile phase composition, temperature,

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humidity, and the size and type of the chamber and its solvent vapor saturation conditions. In the classical method of linear, ascending development TLC and HPTLC, the developing solvent is contained in a large volume, covered glass tank (N-tank). The spotted plate is inclined against an inside wall of the tank with its lower edge immersed in the developing solvent below the starting line, and the solvent begins to rise immediately through the initial zones due to capillary ow. As the mobile phase ascends, the layer interacts with the vapor phase as well as the mobile phase and the mixture components. The space inside the tank is more or less equilibrated with solvent vapors, depending on the presence or absence of a mobile phase-soaked paper liner, and the period of time the tank is allowed to stand before the plate is inserted. Reproducible results are obtained only if all development conditions are maintained as constant as possible. Solvent consumption is high with classical chambers. A sandwich chamber (S-chamber) consists of the TLC plate, a spacer of 3 mm thickness, and a cover- or counterplate that is either blank glass or a solvent-soaked TLC plate. These parts are clamped together so that the bottom 2 cm of the layer is uncovered and are placed in a trough containing the mobile phase. Interaction between the layer, dry or wetted, and the gas phase is largely suppressed in an S-chamber, and reproducibility of the separation is improved. Both ascending and horizontal chambers can be operated as S-chambers (Gocan, 2001a). The twin-trough chamber is an N-chamber modied with an inverted V-shaped ridge on the bottom dividing the tank into two sections, which allow development with only 5 20 mL of solvent, depending on the plate size, on one side, and easy pre-equilibration of the layer with vapors of the mobile phase or another conditioning liquid (e.g., a sulfuric acid water mixture to control humidity) or volatile reagent on the other side. 2. Horizontal Development

or 36 samples from one end to the other. The developing solvent, held in narrow troughs, is carried to the layer through capillary slits formed between the trough walls and the glass slides. The chamber is covered with a glass plate during pre-equilibration and development and can be operated in N-type or S-type congurations, including humidity control by placing an appropriate sulfuric acid water mixture in a conditioning tray. Use of the horizontal developing chamber allows separation conditions to be efciently standardized, and only low amounts of mobile phase are required. The Camag HPTLC Vario System is a horizontal chamber that facilitates development and optimization of separation parameters. Simultaneous development can be tested with up to six different mobile phases, sandwich or tank congurations, and pre-equilibration conditions in any combination. 3. Continuous Development

The short bed continuous development chamber (Regis, Morton Grove, IL) is used for continuous development, which leads to improved resolution of zones with low migration rates because of the larger effective separation distance. Four glass ridges and the back wall of the chamber support the plate at ve different angles of inclination, each of which allows increasing lengths of the layer to protrude out of the top of the chamber. Solvent continually evaporates from the external layer at the top and is replaced by additional solvent drawn from the chamber at the bottom. 4. Gradient Elution TLC Combined with AMD

The horizontal developing chamber (Fig. 5) permits simultaneous development from opposite edges to the middle of 72 sample spots on a 20 10 cm HPTLC plate,

AMD generally involves 10 30 individual linear ascending developments of an HPTLC plate (usually silica gel) carried out in an AMD instrument (Fig. 6), which includes an N-type chamber equipped with connections for feeding and releasing mobile phase and pumping a gas phase in and out, storage bottles for pure solvents and waste, a gradient mixer, syringes for measuring solvent volumes, and a charge coupled device (CCD) detector for monitoring migration distances. The developments are performed in the same direction with a stepwise mobile phase gradient

Figure 5 Horizontal development chamber. 1, HPTLC plate with layer facing down; 2, glass plate for sandwich conguration; 3, reservoir for mobile phase; 4, glass strip; 5, cover plate; 6, conditioning tray. (Courtesy of Camag.)

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Complex mixtures containing compounds with widely different polarities can be separated by AMD on one chromatogram, in which sharply focused zones migrate different distances according to their polarities. Zone widths are independent of migration distance and size of the starting zone, leading to high resolution and the ability to resolve relatively large samples for trace analysis. Migration distances of individual components are largely independent of the sample matrix, and detection limits are improved because of the highly concentrated zones (typically 1 mm peak width) that are produced. The densitometer scan shown in Fig. 7 illustrates a high-resolution chromatogram that can be produced by gradient AMD. 5. Two-Dimensional Development

Figure 6 AMD 2 automated multiple development instrument. (Courtesy of Camag Scientic Inc.)

that becomes progressively weaker (i.e., less polar) over distances that increase by 1 5 mm for each stage. The solvent is completely removed from the chamber, and the layer is dried under vacuum applied by a pump for a preselected time after each development. The layer is then preconditioned with the vapor phase of the next batch of fresh solvent, which is fed into the chamber before the following incremental run. The solvent strength may be changed for each development, or several stages may be carried out with the same solvent before changing its strength. The repeated movement of the solvent front through the chromatographic zones causes them to become compressed into narrow bands during AMD, leading to peak capacities of more than 50 over a separation distance of 80 mm. Typical universal gradients for AMD are produced from methanol or acetonitrile (polar); dichloromethane, di-isopropyl ether, or t-butylmethyl ether (medium polarity); and hexane (nonpolar). The central or base solvent and the nonpolar solvent have the greatest effect on selectivity. By superimposing the densitogram of a chromatogram with a matched-scale diagram of the gradient, required modications of the solvent system can be predicted. Gradient development in TLC has been reviewed (Golkiewicz, 2003).

Two-dimensional (2D) TLC involves spotting the sample in one corner of the layer, developing (ascending or horizontal) with the rst mobile phase, drying the plate, and developing at a 908 angle with a second mobile phase having a diverse, complementary separation mechanism (selectivity). Computer simulation has been used to optimize 2D separations based on one-dimensional (1D) data. Resolution (spot capacity) in 2D TLC is greatly improved compared with 1D TLC because sample components are resolved over the entire area of the layer, and is usually superior to that obtained by HPLC. Resolution by 2D forced ow planar chromatography (FFPC) (see Section VI.B) exceeds 2D capillary-ow TLC because spot diffusion is smaller. Disadvantages of 2D TLC include a limit of one sample per plate and the time required for two developments and intermediate drying. In addition, quantitative calibration or qualitative identication standards cannot be developed in parallel on the same plate at the same time. Improved video densitometers may allow more reliable quantitative 2D TLC in the future than is possible today with the commonly used slit-scanning densitometers. 2D TLC and other multidimensional methods have been reviewed (Gocan, 2001b). B. Forced Flow Planar Chromatography

The mobile phase can migrate through the layer by capillary action, as is the case in the procedures described earlier, or under the inuence of forced ow. FFPC has theoretical advantages relative to capillary ow, including independent optimization of mobile phase velocity, higher efciency, lower separation time, and use of solvents that do not wet the layer, but it requires specialized, complex commercial instrumentation. Forced ow is produced by mechanically pumping solvent through a sealed layer [overpressured layer chromatography or optimum performance laminar chromatography (OPLC) (Mincsovics et al., 2003; Rozylo,

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Analytical Instrumentation Handbook

Figure 7

Multiwavelength densitogram of 16 pesticides separated by AMD. (Courtesy of Camag.)

2001)] or by spinning a glass rotor covered with sorbent around a central axis to drive the solvent from the center to the periphery of the layer by centrifugal force [rotation planar chromatography (RPC)]. Separations can be accomplished with a dry layer (off-line FFPC), but the closed system arrangement also allows the separation to be started after the layer is equilibrated with the mobile phase, similar to the situation in HPLC (on-line FFPC). In RPC, samples are applied to the rotating stationary phase near the center, and centrifugal force along with capillary action drives the mobile phase through the sorbent from the center to the periphery of the plate. Up to 72 samples can be applied for analytical separations, and in situ quantication is possible. One circular sample is applied for micropreparative and preparative separations, which can be carried out off- and on-line. Various chambers are used for RPC, which differ mainly in the volume of the vapor space. The major commercial instruments for RPC are the Chromatotron 7924 (Harrison Research, Palo Alto, CA), CLC-5 (Hitachi, Tokyo, Japan), Extrachrom (RIMP, Budakalasz, Hungary), and Rotachrom Model P (Petazon, Zug, Switzerland). Although analytical applications have been proposed, RPC appears to be most useful for preparative applications. OPLC combines many advantages of classical TLC and HPLC. A special glass- or aluminum-backed layer, sealed at the edges, is covered by a polyethylene or Teon foil and pressurized by water. Development modes include linear unidirectional, linear bidirectional, circular, online, off-line, parallel coupled multilayer, serial coupled multilayer, isocratic, and gradient. The fully automatic,

computer controlled personal OPLC BS-50 instrument (OPLC-NIT, Budapest, Hungary) is shown in Fig. 8 (Mincsovics et al., 1999). It consists, in general, of a separation chamber and a liquid delivery system with a

Figure 8 Automated personal OPLC BS-50 system. 1, Liquid delivery system; 2, separation chamber; 3, cassette; 4, mobile phase inlet; 5, mobile phase outlet; 6, mobile phase switching valve; 7, mobile phase reservoirs; 8, liquid crystal display. [Reproduced from Mincsovics et al. (1999) with permission.]

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two-in-one hydraulic pump and mobile phase delivery pump. The chamber contains a holding unit, hydraulic unit, tray-like layer cassette, and drain valve. The separation chamber has two mobile phase connections and a 5 MPa maximum external pressure.

VII.

ZONE DETECTION

After development with the mobile phase, the plate is dried in a fumehood (with or without heat) to completely evaporate the mobile phase. Separated compounds are detected (or visualized) on the layer by their natural color, natural uorescence, quenching of uorescence, or as colored, UV-absorbing, or uorescent zones after reaction with an appropriate reagent (post-chromatographic derivatization) (Fried and Sherma, 1999h; Sherma, 2001a). Although dependent upon the particular analyte and the detection method chosen, sensitivity values are generally in the low microgram to nanogram range for absorbance and picogram range for uorescence. Layers are frequently heated after applying the detection reagent in order to accelerate the reaction upon which detection is based. Heating is carried out with a hair drier in a fume hood, in an oven, or with a TLC plate heater. The plate heater (Fig. 9), which contains a 20 20 cm at, even heating area, a grid to facilitate proper positioning of TLC and HPTLC plates, programable temperature between 258C and 2008C, and digital display of the actual temperature, provides the most consistent heating conditions. Compounds that are naturally colored are viewed directly on the layer in daylight, whereas compounds with native uorescence are viewed as bright zones on a

dark background under UV light. Viewing cabinets incorporating shortwave (254 nm) and longwave (366 nm) UV lamps are available for inspecting chromatograms in an undarkened room. Compounds that absorb around 254 nm, particularly those with aromatic rings and conjugated double bonds, can be detected on an F-layer containing a phosphor or uorescent indicator (often zinc silicate). When irradiated with 254 nm UV light, absorbing compounds diminish (quench) the uniform layer uorescence and are detected as dark violet spots on a bright (usually green) background. Universal or selective chromogenic and uorogenic liquid detection reagents are applied by spraying or dipping the layer. Various types of aerosol sprayers are available for manual operation, including the Desaga Sprayer SG 1 with a quiet, built-in pump and PTFE spray head (Fig. 10). The ChromaJet DS 20 (Desaga) (Fig. 11) is a spraying instrument that reproducibly applies selectable, accurate amounts of reagent to individual plate tracks under computer control. For safety purposes, spraying is carried out inside a laboratory fumehood or commercial TLC spray cabinet with a blower (fan) and exhaust hose. The most uniform dip application of reagents can be achieved by use of a battery operated chromatogram immersion device (Camag), which provides selectable, consistent vertical immersion and withdrawal speeds between 30 and 50 mm/s and immersion times between 1 and 8 s for plates with 10 or 20 cm heights. This mechanized dipping device can also be used for prewashing TLC plates prior to initial zone application, impregnation of layers with reagents that improve resolution or detection prior to initial zone application and

Figure 9

TLC plate heater. (Courtesy of Camag.)

Figure 10

Sprayer SG 1. (Courtesy of Desaga.)

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Figure 11

ChromaJet DS 20 automatic spray apparatus. (Courtesy of Desaga.)

development, and for postdevelopment impregnation of chromatograms containing uorescent zones with a uorescence enhancer and stabilizer such as parafn. A few detection reagents (HCl, sulfuryl chloride, iodine) can be transferred uniformly to the layer as vapors in a closed chamber. The preparation, procedure for use, and results for many hundreds of TLC detection reagents have been described (Jork, et al., 1990, 1994; Zweig and Sherma, 1972). A variety of biological detection methods are available for compound detection. As an example, immunostaining of thin layer chromatograms provides very sensitive detection of glycolipids (Putalan et al., 2001).

VIII.

DOCUMENTATION OF CHROMATOGRAMS

TLC plates contain complete chromatograms that provide a great amount of information for sample identication, visual semiquantication, and comparison, especially when different detection methods are used to give multiple images of the same sample. TLC separations are documented by photography, video recording, or scanning (Fried and Sherma, 1999i; Morlock and Kovar, 2003). Commercial systems for photographic documentation contain a conventional, instant, or digital (Fig. 12) camera and associated lighting accessories for photography of colored, uorescent, and uorescence-quenched zones on TLC plates in shortwave, midrange, and longwave UV light and in visible light. Special software is needed for reproducible, GMP/GLPcompliant use of a digital camera. The Camag video documentation system (VideoStore, Fig. 13) includes visible and UV lighting, CCD camera with zoom lens, monitor, and video color printer. The

Figure 12 Reprostar 3 lighting unit and camera stand with cabinet cover and mounted digital camera. (Courtesy of Camag.)

VideoStore software is GMP/GLP-compliant for capture, editing, annotation, documenting, and archiving of images. Desaga offers a similar color video documentation system (VD 40) with eightfold motor zoom and autofocus CCD camera, pentium powered computer, and ProViDoc GLP-conforming software. Documentation can also be carried out by scanning the spots on the plate with a atbed ofce scanner (Rozylo

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IX.

ZONE IDENTIFICATION

Figure 13 Camag VideoStore/VideoScan including Reprostar 3 with cabinet cover, camera bellows, camera support, and 3-CCD camera with zoom objective. (Courtesy of Camag.)

et al., 1997). The equipment required includes a computer, scanner, and monochrome or color printer. Computer scanning can be used only for visible spots, but not those that are uorescent or quench uorescence unless the atbed scanner is modied.

The identity of TLC zones (Fried and Sherma, 1999i; Morlock and Kovar, 2003) is obtained in the rst instance by comparison of Rf values between samples and reference standards chromatographed on the same plate, where Rf equals the migration distance from the origin to the center of the zone divided by the migration distance of the mobile phase front. Identity is more certain if a selective chromogenic reagent yields the same characteristic color for sample and standard zones, or if an Rf match between samples and standards is obtained in at least two TLC systems with diverse mechanisms, for example, silica gel NP and C-18 bonded silica gel RP. Comparison of standard and sample in situ UV-visible absorption or uorescence emission spectra, obtained by using the spectral mode of a slit-scanning or video densitometer, can also aid identication, but these spectra may contain inadequate structural information for complex mixtures. The TIDAS TLC 2010 scanner (Flowspek AG, Basel, Switzerland) (Fig. 14) allows rapid scanning of TLC plates with simultaneous acquisition of a complete spectrum for all substances on a layer. Fiber optics technology is combined with diode array detection in this instrument, which has the following specications: 190 1000 nm wavelength range, 0.8 nm pixel resolution, deuterium and tungsten light sources, ,160 mm optical resolution

Figure 14 TLC 2010 diode array scanner. (Courtesy of Flowspek.)

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Figure 15 Contour plot, densitogram, and codeine spectrum on one screen shot, with codeine appearing at 13.6 mm. (Courtesy of Flowspek.)

on the layer, 5 mm/s scanning speed, 0.1 mm positioning accuracy, and software with parameters including peak purity, resolution, and identication via spectral library matching. Figures 15 and 16 show the identication and conrmation of codeine in urine with the TLC 2010 diode array scanner. Zone identity can also be conrmed by the application of combined TLC-spectrometry methods described in Section XI.

X. A.

QUANTITATIVE ANALYSIS Nondensitometric Methods

Figure 16 Codeine spectrum (top) and library spectrum (bottom) with a match of 98%. (Courtesy of Flowspek.)

Quantication of thin layer chromatograms (Fried and Sherma, 1999j; Prosek and Vovk, 2003) can be performed after manually scraping off the separated zones of samples and standards and elution of the substances from the layer material with a strong, volatile solvent. The eluates are concentrated and analyzed by use of a spectrometry, GC, HPLC, or some other sensitive microanalytical method. This method of quantication is laborious and time consuming, and the difculty in recovering samples and standards uniformly is a major source of error. Although its importance has declined relative to densitometry, the indirect scraping and elution quantication method is still being rather widely used, for example, for some drug assays according to the US Pharmacopoeia. Direct TLC semiquantitative analysis can be performed by visual comparison of sample spot intensities with the intensities of reference spots developed simultaneously on the same layer. For this comparison, the bracketing method is used in which standard spots with concentrations equal to, greater than, and less than the expected sample concentration are placed on either side of duplicate sample spots. The concentrations of samples and standards should lie within the linear response range of the detection method. The use of TLC for compliance screening of drug products is an important example of this approach.

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B.

Densitometric Evaluation

Most modern HPTLC quantitative analyses are performed by in situ measurement of the absorbance or uorescence of the separated zones in the chromatogram tracks using an optical densitometic scanner (Reich, 2003; Sherma, 2001b) operated with a xed sample light beam in the form of a rectangular slit. The length and width of the slit is selectable for optimized scanning of spot or bandshaped zones with different dimensions. The densitometer measures the difference between the optical signal from a zone-free background area of the plate and that from the calibration standards and sample zones. With automated zone application, precision ranging from 1% to 3% RSD is typical for densitometric analyses. The plate is mounted on a moveable stage controlled in the X- and Y-directions by a stepping motor, which allows each chromatogram to be scanned, usually in the direction of development. The single beam, single wavelength scanning mode most often used gives excellent results with high quality plates and a mobile phase that result in compact, well separated zones. A schematic diagram of the light path of a densitometer is shown in Fig. 17. A tungsten-halogen lamp is used as the source for scanning

colored zones in the 400 800 nm range (visible absorption) and a deuterium continuum lamp for scanning the absorption of UV light by zones on layers with or without uorescence indicator in the 190 400 nm range. The monochromator used with these continuous wavelength sources can be a quartz prism or, more often, a grating. The detector is a photomultiplier or a photodiode. For uorescence scanning, a high intensity xenon or mercury vapor lamp is used as the source, the optimum excitation wavelength is selected by the monochromator, and a cutoff lter is placed between the plate and detector to block the exciting UV radiation and transmit the visible emitted uorescence. The light beam strikes the plate at a 908 angle, and the photomultiplier for reectance scanning is at a 30o angle to normal. Part of the light beam is directed to a reference photomultiplier by a beam splitter to compensate for lamp variations and short-term uctuations. A detector mounted below the stage is used when scanning in the transmission mode (TLC plates or electrophoresis gels). Zig-zag (or meander) scanning with a small spot of light is possible with scanners having two independent stepping motors to move the plate in the x- and y-axes. Computer algorithms integrate the maximum absorbance measurements from each swing, which

Figure 17 Light path diagram of the TLC Scanner 3. 1, Lamp selector; 2, entrance lens slit; 3, monochromator entry slit; 4, grating monochromator; 5, mirror; 6, slit aperture disk; 7, lens system; 8, mirror; 9, beam splitter; 10, reectance monochromator; 11, object to be scanned; 12, measuring photomultiplier; 13, photodiode (transmission). [Reproduced from Reich (2003) with permission.]

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corresponds to the length of the slit, to produce a distribution prole of zones having any shape. Disadvantages of scanning with a moving light spot include problems with data processing, lower spatial resolution for HPTLC, and unfavorable error propagation upon averaging of readings from different points within the zone. Most modern scanners have a computer controlled motor driven monochromator that allows automatic recording of in situ absorption and uorescence excitation spectra at multiple wavelengths (Fig. 7). These spectra can aid compound identication by comparison with cochromatographed standards or stored standard spectra, test for identity by superimposition of spectra from different zones on a plate, and check zone purity by superimposition of spectra from different areas of a single zone. The spectral maximum determined from the in situ spectrum is usually the optimal wavelength for scanning standard and sample areas for quantitative analysis. The scanner is connected to a recorder, an integrator, or a computer. A personal computer with software designed specically for TLC is most common for data processing and automated control of the scanning process in modern instruments. With a fully automated system (e.g., winCATS from Camag), the computer can carry out the following functions: selectable scanning speed up to 100 mm/s; evaluation of 36 tracks with up to 100 substances in sequence; integration with automatic or manual baseline correction; single or multilevel calibration with linear or nonlinear regression using internal or external standards, and statistics such as RSD or condence interval with full error propagation; subcomponent evaluation to relate unidentied fractions to the main component, as required by various pharmacopoeias; dual-wavelength scan to eliminate matrix effects or quantify incompletely resolved peaks; multiwavelength scan (up to 31 different wavelengths) to obtain the optimum wavelength for quantication of each fraction and achieve maximum analytical selectivity; scanner qualication (automatic tests of mechanical, optical, and electronic functions); track optimization (repeated scanning of each track with small lateral offsets in order to optimize measurements of distorted chromatograms); and spectrum library. For GMP compliance, all conditions and data are automatically recorded and held in a secure format. Because of light scattering from the sorbent particles, a simple, well-dened mathematical relationship between amount of analyte and the light signal has not been found. Plots relating absorption signal (peak height or area) and concentration or weight of standards on the layer are usually nonlinear, especially at higher concentrations, and do not pass through the origin. Modern integrators and computer software programs can routinely perform linear or polynomial regression of the calibration data, depending upon which is most suitable. Fluorescence

calibration curves are generally linear and pass through the origin, and analyses based on uorescence are more specic and 10 1000 times more sensitive than those employing absorbance. Because of these advantages, compounds that are not naturally uorescent are often derivatized pre- or postchromatography to allow them to be scanned in the uorescence mode if an appropriate reagent is available. However, absorbance in the 190300 nm UV range has been most used for densitometric analyses. Validation procedures (Fried and Sherma, 1999k) for quantitative analysis are in some aspects very similar to those for HPLC and GC, with additional considerations related to procedural aspects specic to TLC. Protocols for validation of TLC results (Ferenczi-Fodor et al., 2001), especially for pharmaceutical analysis, are available in the literature. Some densitometers include automatic instrument validation programs in their software. The Camag TLC Scanner 3, Shimadzu (Columbia, MD USA) CS 9000, and Desaga Densitometer CD 60 are examples of modern computer-controlled slit-scanning densitometers. Video densitometers (image processors) are an alternative to the optical/mechanical slit scanners. Video densitometry is based on electronic point-scanning of a stationary plate using an instrument composed of UV and visible light sources, a CCD camera with zoom capabilities, and a computer with imaging and evaluation software. Video scanners have advantages including rapid data collection and storage, simple design with virtually no moving parts, easy operation, and the ability to quantify 2D chromatograms, but they have not yet been shown to have the required capabilities to replace slit-scanning densitometers. Current video scanners can function only in the visible range to measure colored, uorescencequenched, or uorescent spots. They lack the spectral selectivity and accuracy based on the ability to scan with monochromatic light of selectable wavelength throughout the visible and UV range (190 800 nm) that are inherent in classical densitometry, and they cannot record the in situ spectra. The VideoScan software program (Camag) allows quantitative evaluation (videodensitometry) of images at any time after they are captured with the VideoStore documentation system shown in Fig. 13. Integration of analog curves can be performed automatically, quantication performed via peak areas or heights, and single or multilevel calibrations with linear or polynomial regression. The ProResult software package is available for quantication with Desagas video documentation system. Special software packages have been used for quantication of zones on chromatogram images produced with a atbed scanner. Both colored (visible) (Johnson, 2000) and uorescent (Stroka et al., 2001) zones have been

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measured for the analyses, the latter after modication of the scanner by adding a black light tube. XI. A. TLC COMBINED WITH SPECTROMETRIC METHODS Mass Spectrometry

The identity of TLC zones can be conrmed by mass spectrometry (MS) analysis (Busch, 2003; Rozylo, 2001). The most used ionization modes for TLC/MS include electrospray ionization (ESI), and matrix- and surface-assisted laser desorption ionization (MALDI and SALDI). ESI is used with solvent extracts of zones scraped from the TLC plate. The liquid is sprayed through a charged needle as an aerosol mist at atmospheric pressure, and the ions created from desolvation and charge distribution processes in the droplets are extracted through skimmer cones into the mass analyzer of the mass spectrometer. MALDI and SALDI involve ionization directly from the surface layer held under vacuum after addition of an energy-buffering matrix. The ionization occurs as a result of surface irradiation by a laser beam, with mass analysis usually carried with a time of ight (TOF) mass analyzer. On-line TLC/ESI MS has been carried out by directly linking an OPLC 50 instrument (Fig. 8) and a Micromass (Beverly, MA USA) Q-TOF mass spectrometer (Chai et al., 2003). Aluminum-backed silica gel layers with a perimeter seal were used. After initial layer preconditioning to reduce background noise, the limit of detection of glycolipids was in the 5 20 pmol range. Quadrupole, ion trap, and Fourier Transform (FT) mass spectrometers and fast atom bombardment and liquid secondary ion mass spectrometry ionization techniques have also been used in various applications of TLC/MS. B. Infrared Spectrometry

the large active surface area of silica gel, with its hydroxyl groups, makes adequate compensation of sample and reference spectra more difcult. It has been found that HPTLC plates with 10 mm particles, small particle size distribution, 0.2 mm layer thickness, and glass backing are best for direct TLC DRIFT. A Brucker IFS 48 FTIR spectrometer with a special mirror arrangement and MCT (mercury, cadmium, telluride) small band detector has been constructed to enable DRIFT measurement despite the self-absorbance of TLC sorbents (Glauninger et al., 1990). Although mainly useful for qualitative identication and conrmation of zones, quantication by TLC IR has been carried out in some applications by evaluation of Kubelka Munk spectra with integration of their strongest bands, especially for substances lacking chromophores that absorb in the UV-visible range. However, the limit of determination is about 10 times higher than that of densitometry, and precision is poorer. C. Raman Spectrometry

Zones can also be conrmed by combining TLC with infrared (IR) spectrometry (Morlock and Kovar, 2003). Indirect TLC IR coupling is carried out by transfer of the sample from the layer to an IR-transparent pellet or powder such as KBr or in situ measurement of scraped TLC zones by the diffuse reectance infrared Fourier transform spectrometry (DRIFT) technique. TLC has been directly coupled with DRIFT, in which case difculties occur because conventional stationary phases, for example, silica gel, absorb strongly in the IR region, and the inuence of the refractive index on the spectra must be considered. Intense interference bands between 1350 and 1000 cm21 and above 3550 cm21 are superimposed on the DRIFT spectra of the analytes and restrict the available wavenumber range. In addition,

Surface-enhanced Raman scattering (SERS) spectrometry (Morlock and Kovar, 2003; Somsen et al., 1995) has sensitivity in the nanogram or the picogram range using a Raman spectrometer with argon ion, HeNe, or YAG monochromatic light source and CCD detector. The method is most useful for identication of compounds with groups of atoms that are IR-inactive; quantitative analysis has not been successfully carried out. For in situ analysis, the HPTLC plate, after development and drying, is dipped into or sprayed with a colloidal silver suspension prepared by reduction of silver nitrate with sodium citrate. Alternatively, the silver molecules can be evaporated onto the layer in order to eliminate the zone diffusion and lowering of enhancement caused by dipping or spraying. Highly Raman-active substances (dyes, optical brighteners) can be determined at low nanogram levels without surface-enhanced scattering on specially modied silica gel plates. EMD chemicals Inc. (Gibbstown, NJ, an afliate of Merck KGaA) sells silica gel 60 plates with a 0.1 mm layer of 3 5 mm particles on an aluminum support. The spherical silica gel produces a 10-fold increase in Raman spectrometry signal intensity compared with a similar layer made with irregular silica gel particles. XII. PREPARATIVE LAYER CHROMATOGRAPHY

PLC for isolation of larger amounts of material than normally separated by TLC can be carried out by classical PLC (Fried and Sherma, 1999l) by use of thicker layers

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(usually 0.5 2 mm) developed in the ascending direction in a large volume chamber with detection of zones by a nondestructive method such as iodine vapor or uorescence quenching. Fractions are scraped from the layer, and the puried analytes recovered by elution with a solvent for use in other laboratory work or further analysis. The construction and use of the commercial instruments available for forced ow PLC have been described by Nyiredy (Nyiredy, 2003). These include the older Chrompres 10 and 25 chambers (LABOR Instrument Works, Budapest, Hungary) and Personal OPLC 50 system (Section VI.B, capable of semipreparative separations only) for OPLC and the Chromatotron CLC-5, Rotachrom, and Extachrom for RPC. An additional commercial instrument for rotation or centrifugal preparative planar chromatography is the CycloGraph II from Analtech (Newark, DE; Fig. 18). Separations typically occur within 20 min without need to scrape off the separated zones. The sample solution is applied using a solvent pump or hand-held syringe inside the adsorbent ring of a precast rotor. The eluent (mobile phase) is pumped through the rotating layer (variable speed control from 100 to 1400 rpm), and as each separated ring reaches the outer rim of the rotor it is spun off of the edge of the glass into a circular trough. The adjustable angle of the trough allows the eluent to settle at the bottom and drip out of the collection port into individual

tubes for different fractions. High performance, reusable silica gel rotors (9.5 in diameter) with 1000 8000 mm thickness are available.

XIII. THIN LAYER RADIOCHROMATOGRAPHY Location and quantication of separated radioisotopelabeled substances on a thin layer requires the use of contact autoradiography, zonal analysis, or direct scanning with a radiation detector. These thin layer radiochromatography (TLRC) methods (Fried and Sherma, 1999m; Hazai and Klebovich, 2003) are especially important in drug and pesticide metabolism studies in plants, animals, and humans and in studies of the fate of labeled chemicals in the environment. Contact autoradiography involves exposure of X-ray or photographic lm to emissions from radioisotope zones to produce an image on the lm. After exposure and development of the lm, the radioisotopes are visible as dark spots, which can be compared with standards for qualitative identication or quantied by measurement of their optical densities with a scanning densitometer. Zonal analysis involves scraping radioactive zones from the plate, placing the sorbent in counting vials, adding scintillation uid or cocktail to elute the radioactive components, and liquid scintillation counting. Direct measurement of radioactive zones on a layer is most often carried out today by use of a linear analyzer, digital autoradiograph (DAR), or an imaging analyzer. Linear analyzers incorporate a position-sensitive windowless gas-ow proportional counter as the detector, which measures all radioactive zones in a chromatogram (track) simultaneously and then is moved under computer control for measurements at other selected positions across the layer. The ll gas (e.g., argon methane) is ionized when radioactive emissions from the TLC zones enter the detector, producing electrons. The electron pulses are detected electronically and stored in computer memory to provide a digital image of the distribution of radioactivity on the layer. An example of a TLC linear analyzer is the RITA (Raytest, Wilmington, NC; Fig. 19), which has a gold plated detector with an active length of 200 mm and an active width selectable from 20 to 1 mm by choice of the diaphragm. Multiwire proportional counters (DAR or microchannel array detector) are 2D detectors based on a measuring principle similar to the linear analyzer, but they can detect all areas of radiation from a 20 20 cm layer simultaneously without moving a detector head. Many research studies have been reported on applications of HPTLC and OPLC coupled with an LB 287 Berthold DAR (EG&G Berthold, Wilbad, Germany) for analysis of tritiated or 14C-labeled metabolites in a variety of

Figure 18 CycloGraph II centrifugal preparative planar chromatography instrument with built-in UV lamp and hinged lid. (Courtesy of Analtech.)

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Figure 19 RITA radioactivity intelligent thin layer analyzer. (Courtesy of Raytest.)

biological matrixes. This instrument has a 20 20 cm sensitive area that contains a 600 600 wire grid. Measurements are made using argon methane (9:1) as counting gas bubbled through methylal at 2.88C and a ow rate of 5 mL/min. The positive high voltage potential used for 3H- and 14C-labeled compounds is 2040 and 1200 V, respectively, and signal analysis is achieved by measuring 5 360,000 detector cells/s. Bioimaging/phosphor imaging analyzers represent the newest and most advantageous technology for measuring radioactive TLC zones. The layer is exposed to a phosphor imaging plate (IP) that accumulates and stores irradiating radioactive energy from the zones. The plate is then inserted into an image-reading unit and scanned with a ne laser beam. Luminescence is emitted in proportion to the intensity of the recorded radiation, collected by a photomultiplier tube, and converted to electrical energy to produce the instrumental readout. Resolution, linear

range, and sensitivity are equal to, or better than, those of the other detection methods. The BAS 5000 (Raytest) (Fig. 20) is an example of a modern phosphor IP TLC scanner. Specications include 20 25 cm IP size, 25/ 50 mm pixel size, 5 min (50 mm) reading time, detection limit 0.9 dpm/mm2/h (14C), and four to ve orders of magnitude (16 bits) dynamic range.

XIV.

APPLICATIONS OF TLC

TLC can provide rapid, low cost qualitative analyses and screening in order to obtain information such as sample stability, purity, and uniformity and to follow the course of a reaction, whereas instrumental HPTLC can provide accurate and reproducible (1 3% RSD) quantitative results. Samples that are difcult to prepare can be analyzed readily, and detection is especially exible in the

Figure 20

BAS 5000 phosphor IP scanner. (Courtesy of Raytest.)

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1012 Table 1

Analytical Instrumentation Handbook Sources of Information on the TLC Analysis of Different Compound Classes Fried and Sherma (1999n) Amino acids Antibiotics Carbohydrates Carboxylic acids Ceramides Coumarins Dyes Enantiomers Indoles Inorganics Lipids Nucleic acid derivatives Organometallics Peptides and proteins Pesticides Pharmaceuticals and drugs Phenols Pigments Plant extracts Steroids Taxoids Terpenoids Toxins Vitamins Sherma and Fried (2003) Cazes (2001)

Note: indicates the book contains a chapter on the compound class.

absence of the mobile phase and with a variety of parameters. TLC has been applied virtually in all areas of analysis, including chemistry, biochemistry, biology, industrial, agricultural, environmental, food, pharmaceutical, clinical, natural products, toxicology, forensics, plant science, bacteriology, parasitology, and entomology. Table 1 lists the compound classes that are covered in three books that contain detailed information on specic applications of TLC analysis, discussion of which is beyond the scope of this chapter. The applications of TLC, as well as theory, techniques, and instrumentation, is regularly updated in a biennial review of planar chromatography (Sherma, 2004) and the Camag Bibliography Service, which is published every March and September and is available in paper and CD-ROM format free of charge.

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Abjean, J. P. (1993). Screening of drug residues in food of animal origin by planar chromatography: application to sulfonamides. J. Planar Chromatogr.-Mod. TLC 6(2):147 148.

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Thin Layer Chromatography Fried, B., Sherma, J. (1999a). Introduction and history. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 1 8. Fried, B., Sherma, J. (1999b). Mechanism and theory. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 9 24. Fried, B., Sherma, J. (1999c). Obtaining material for TLC and sample preparation. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 51 76. Fried, B., Sherma, J. (1999d). Sorbents, layers, and precoated plates. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 25 49. Fried, B., Sherma, J. (1999e). Solvent systems. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 89 107. Fried, B., Sherma, J. (1999f). Application of samples. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 77 87. Fried, B., Sherma, J. (1999g). Development techniques. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 109 144. Fried, B., Sherma, J. (1999h). Detection and visualization. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 145 175. Fried, B., Sherma, J. (1999i). Qualitative identication and documentation. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 177 196. Fried, B., Sherma, J. (1999j). Quantication. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 197 222. Fried, B., Sherma, J. (1999k). Reproducibility and validation of results. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 223 233. Fried, B., Sherma, J. (1999l). Preparative layer chromatography. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 235 248. Fried, B., Sherma, J. (1999m). Radiochemical techniques. Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc., pp. 249 267. Fried, B., Sherma, J. (1999n). Thin Layer Chromatography. 4th Ed. New York: Marcel Dekker, Inc. Glauninger, G., Kovar, K. A., Hoffmann, V. (1990). Possibilities and limits of an online coupling of thin layer chromatography and FTIR spectroscopy. Fresenius J. Anal. Chem. 338(6):710 716. Gocan, S. (2001a). TLC sandwich chambers. In: Cazes, J., Ed. Encyclopedia of Chromatography. New York: Marcel Dekker, Inc., pp. 851 854. Gocan, S. (2001b). Multidimensional TLC. In: Cazes, J., Ed. Encyclopedia of Chromatography. New York: Marcel Dekker, Inc., pp. 533 535. Golkiewicz, W. (2003). Gradient development in thin layer chromatography. In: Sherma, J., Fried, B., eds. Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc., pp. 153 173. Hamada, M., Wintersteiger, R. (2002). Determination of phenylurea herbicides in drinking water. J. Planar Chromatogr.Mod. TLC 15(1):11 18.

1013 Hauck, H.-E., Schulz, M., Schaefer, C. (2002). Ultra-thin-layer chromatography (UTLC): the new dimension in planar chromatography. In: Vovk, I., Medja, A., eds. Proceedings of the International Symposium Planar Chromatography Today 2002. Ljubljana, Slovenia: National Institute of Chemistry, pp. 61 66. Hazai, I., Klebovich, I. (2003). Thin layer radiochromatography. In: Sherma, J., Fried, B., eds. Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc., pp. 339 360. Johnson, M. E. (2000). Rapid, simple quantitation in thin layer chromatography using a atbed scanner. J. Chem. Educ. 77(3):368 372. Jork, H., Funk, W., Fischer, W., Wimmer, H. (1990). Thin Layer Chromatography, Volume 1a, Physical and Chemical Detection Methods. Weinheim, Germany: VCH Verlagsgesellschaft mbH. Jork, H., Funk, W., Fischer, W., Wimmer, H. (1994). Thin Layer Chromatography, Volume 1b, Reagents and Detection Methods. Weinheim, Germany: VCH Verlagsgesellschaft mbH. Kowalska, T., Prus, W. (2001). Theory and mechanism of thin layer chromatography. In: Cazes, J., Ed. Encyclopedia of Chromatography. New York: Marcel Dekker, Inc., pp. 821 825. Kowalska, T., Kaczmarski, K., Prus, W. (2003). Theory and mechanism of thin layer chromatography. In: Sherma, J., Fried, B., eds. Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc., pp. 4780. Lepri, L., Cincinelli, A. (2001). TLC sorbents. In: Cazes, J., Ed. Encyclopedia of Chromatography. New York: Marcel Dekker, Inc., pp. 854 858. Mincsovics, E., Garami, M., Kecskes, L., Tapa, B., Katy, G., Tyihak, E. (1999). Personal overpressured layer chromatography (OPLC) basic system 50, exible tool in analytical and semipreparative work. J. AOAC Int. 82(3):587 598. Mincsovics, E., Ferenczi-Fodor, K., Tyihak, E. (2003). Overpressured layer chromatography. In: Sherma, J., Fried, B., eds. Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc., pp. 175 205. Morlock, G., Kovar, K.-A. (2003). Detection, identication, and documentation. In: Sherma, J., Fried, B., eds. Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc., pp. 207 238. Nurok, D., Koers, J. M., Carmichael, M. A., Liao, W.-M., Dzido, T. H. (2002). The performance of planar electrochromatography in a horizontal chamber. J. Planar Chromatogr.Mod. TLC 15(5):320 323. Nyiredy, Sz. (2003). Preparative layer chromatography. In: Sherma, J., Fried, B., eds. Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc., pp. 307 338. Prosek, M., Vovk, I. (2003). Basic principles of optical quantication in TLC. In: Sherma, J., Fried, B., eds. Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc., pp. 277 306. Prus, W., Kowalska, T. (2001). Optimization of thin layer chromatography. In: Cazes, J., Ed. Encyclopedia of

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1014 Chromatography. New York: Marcel Dekker, Inc., pp. 576 579. Putalan, W., Tanaka, H., Shoyama, Y. (2001). TLC immunostaining of steroidal alkaloid glycosides. In: Cazes, J., Ed. Encyclopedia of Chromatography. New York: Marcel Dekker, Inc., pp. 849 851. Rabel, F. J. (2003). Sorbents and precoated layers in thin layer chromatography. In: Sherma, J., Fried, B., eds. Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc., pp. 99 134. Reich, E. (2003). Instrumental thin layer chromatography (planar chromatography). In: Sherma, J., Fried, B., eds. Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc., pp. 135 151. Rozylo, J. K. (2001). Overpressured layer chromatography. In: Cazes, J., Ed. Encyclopedia of Chromatography. New York: Marcel Dekker, Inc., pp. 579582. Rozylo, J. K., Siembida, R., Jamrozek-Manko, A. (1997). A new simple method for documentation of TLC plates. J. Planar Chromatogr.-Mod. TLC 10(3):225 228. Rozylo, J. K. (2001). Thin layer chromatography mass spectrometry. In: Cazes, J., Ed. Encyclopedia of Chromatography. New York: Marcel Dekker, Inc., pp. 839842. Sherma, J. (2001a). Detection (visualization) of TLC zones. In: Cazes, J., Ed. Encyclopedia of Chromatography. New York: Marcel Dekker, Inc., pp. 248251.

Analytical Instrumentation Handbook Sherma, J. (2001b). Optical quantication (densitometry) in TLC. In: Cazes, J., Ed. Encyclopedia of Chromatography. New York: Marcel Dekker, Inc., pp. 572 576. Sherma, J. (2002a). Thin layer chromatography. In: Issaq, H. J., Ed. A Century of Separation Science. New York: Marcel Dekker, Inc., pp. 49 68. Sherma, J. (2003). Basic techniques, materials, and apparatus. In: Sherma, J., Fried, B., eds. Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc., pp. 1 46. Sherma, J., Fried, B., eds. (2003). Handbook of Thin Layer Chromatography. 3rd Ed. New York: Marcel Dekker, Inc. Sherma, J. (2004). Planar chromatography. Anal. Chem. 76(12):3251 3262 (Previous biennial reviews of TLC by J. Sherma are contained in issue 12 of this journal each even numbered year dating back to volume 42. 1970.). Somsen, G. W., Morden, W., Wilson, I. D. (1995). Planar chromatography coupled with spectroscopic techniques. J. Chromatogr. A 703(1 2):613 665. Stroka, J., Peschel, T., Tittelbach, G., Weidner, G., van Otterdijk, R., Anklam, E. (2001). Modication of an ofce scanner for the determination of aatoxins after TLC separation. J. Planar Chromatogr.-Mod. TLC 14(2):109 112. Zweig, G., Sherma, J. (1972). Handbook of Chromatography, Volume II. Boca Raton, FL: CRC Press.

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